Preparation of Phosphoinositide-Containing Supported Lipid Bilayers and Quantitative Assessment of Protein Recruitment to Membrane Surfaces

Materials and reagents

Reagents

Liposomes

1. Chloroform (Sigma-Aldrich, catalog number: 366927-1L)

2. Argon gas

3. HEPES [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid], 1 M, pH 7.8 (Sigma-Aldrich, catalog number: H4034)

4. NaCl, 5 M (Euromedex, catalog number: 1112-A)

5. Phosphatidylcholine (Avanti, catalog number: 840054P-200mg, soy)

6. Phosphatidylethanolamine (Avanti, catalog number: 840022P-25mg, brain, porcine)

7. Phosphatidylserine (Avanti, catalog number: 840032P-25mg, brain, porcine)

8. Cholesterol (Avanti, catalog number: 700000-100mg, ovine)

9. NBD-PE [N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) phosphoethanolamine] (Avanti, catalog number: 840145P-1mg, 18:1)

10. Phosphatidylinositol-3,4-bisphosphate (Avanti, catalog number: 850153P-100μg, 18:1)

11. DGS-NiNTA (1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] (nickel salt) (Avanti, catalog number: A89404, 5mg, 18:1)

PDMS support

1. Sylgard 184 silicone elastomer (Sigma-Aldrich, catalog number: 761036-5EA)

2. Ethanol (≥99.8%) (VWR Chemicals, PROLABO, catalog number: 20821296-1L)

3. Isopropanol (≥99.8%) (Fluka, catalog number: 34965-1L)

4. Deionized water and MilliQ water

5. Absolute acetone (≥99.8%) (Merck, catalog number: 100014)

6. HMDS (hexamethyldisilazane) (Sigma-Aldrich, catalog number: 440191-100ML)

SLB

1. CaCl₂, 1 M (Merck, catalog number: 10035-04-8)

TIRF imaging

1. Immersion oil (e.g., ZEISS, catalog number: 444960 518F)

Solutions

1. Lipid stock solutions in chloroform (see Recipes)

2. HN buffer (see Recipes)

3. SLB-FB buffer (see Recipes)

4. HEPES 20 mM (pH 7.5) (see Recipes)

Recipes

1. Lipid stock solutions in chloroform

We provide an example of the lipid composition used in the experiments presented in this protocol.

See the table below to prepare 0.5 mL of lipid stock solutions at a final lipid concentration of 2.0 mM (thereafter, “2 mM liposome”).

Lipid stock solutions to prepare 0.5 mL at 2 mM liposome

2. HN buffer

HEPES 20 mM (pH 7.5), NaCl 100 mM

3. SLB-FB buffer

HEPES 20 mM (pH 7.5), NaCl 120 mM, DTT 1 mM

4. HEPES 20 mM (pH 7.5)

NaCl 120 mM, DTT 1 mM, MgCl₂ 1 mM

Laboratory supplies

Liposomes

1. 1.5 mL opaque tubes (e.g., Eppendorf, catalog number: 0030120191) and Amber tubes (Eppendorf, catalog number: 10458312)

2. Glass vials (e.g., WHEATON^®, catalog number: DWKW224681) (2 mL)

3. Powder-free nitrile gloves (e.g., SHIELD Scientific, catalog number: 625124)

4. Quickfit^® round-bottom flasks (e.g., Sigma-Aldrich, catalog number: Z302732-1EA)

PDMS support

1. Silicon master molds (photolithography generated) (homemade)

2. Aluminum foil (e.g., Sigma-Aldrich, catalog number: Z691577)

3. Adhesive tape (e.g., Scotch 3M)

4. Glass coverslips, e.g., 24 × 60 mm with 0.13–0.17 mm thickness (VWR, catalog number: 48404-133)

5. 6-mm biopsy punch (e.g., Dutscher, catalog number: 030740)

SLB

1. Glass coverslip holder

Equipment

Liposomes

1. Rotavapor (e.g., Labortechnik AG, model: eR-II BÜCHI)

2. Vacuum pump (e.g., Büchi Labortechnik AG, model: V-700 with vacuum controller V-850)

3. Liquid cooling system (e.g., Fisher Bioblock Scientific)

4. Vortex mixer (e.g., Sigma-Aldrich, catalog number: Z258423-1EA)

5. Hamilton^® syringes [e.g., Sigma-Aldrich, catalog numbers: HAM80075-1EA (10 μL); HAM7656-01-1EA (100 μL); 20739 (500 μL)]

PDMS support

1. Ultrasonic bath (e.g., Bandelin SONOREX, Merck, catalog number: Z659460)

2. Vacuum desiccator or vacuum chamber

3. Oven (75 °C)

4. Plasma cleaner (e.g., Harrick, catalog number: PDC-32G-2, 230V)

5. Closed chamber/container for HMDS vapor silanization

6. Chemical hood

TIRF imaging

1. Cyanine5.5 succinimidyl ester (CY5.5-SE) (e.g., Cytiva, catalog number: 10461835)

2. TIRF microscope, e.g., Nikon Ti-E inverted equipped with a 100× TIRF Nikon objective (NA 1.49, oil immersion) and a 1.5× Nikon magnification lens. Fluorescence excitation is achieved using 488, 561, and 640 nm lasers: 488 nm Coherent OBIS laser 150 mW for NBD-PE and GFP-derived probes; 561 nm Coherent OBIS laser 100 mW Cy3 protein fluorophores; 640 nm Coherent OBIS laser 150 mW for Cy5 or Cy5.5-labeled proteins. Excitation light and fluorescence emission are separated using a quad-band dichroic filter (TRF89901v2-NK, Chroma), and the fluorescence signal will be detected on an EMCCD camera (iXion Ultra 897, Andor, pixel size = 107 nm). Hardware control and image acquisition are performed using MetaMorph (Molecular Devices, v7.8.13.0)

3. Centrifuge (e.g., Sigma-Aldrich, model: Eppendorf EP5810000010-1EA)

4. Vacuum pump (e.g., Lagerwerk, model: KNF N86 KN.18)

Software and datasets

Image acquisition and TIRF illumination were controlled using MetaMorph Microscopy Automation and Image Analysis Software (Molecular Devices, San Jose, CA, USA; RRID: SCR_002368) and iLAS2/Modular TIRF control software (Gataca Systems, Massy, France). Quantification of membrane-associated fluorescence was performed with Fiji software [19] and fitting of raw curves and statistical analysis with PRISM 10 (GraphPad).

Procedure